The overall goal of this project is to develop a framework for understanding the way in which membrane proteins expressed on pathogen cell membranes mediate the interactions of pathogens with their human host. We focus on Ail (attachment invasion locus), a protein expressed in the outer cell membrane of the bacterium Yersinia pestis, which is the causative agent of plague. The Y. pestis bacterium is highly pathogenic, spreads rapidly and causes extremely high human mortality. Although it is sensitive to a restricted panel of antibiotics, the potential weaponization of aerolized bacteria with bio-engineered antibiotic resistance and the lack of an effective vaccine or therapy are major concerns, contributing to its classification as a Tier 1 Biothreat Agent. Ail is a key bacterial virulence factor and a prime candidate for therapeutic development due to its functions in mediating bacterial adhesion to host cells, and promoting bacterial resistance to human immunity. This project focuses on elucidating the molecular mechanisms of these key functions of Ail by determining its three-dimensional structure and structurally characterizing the interactions of Ail with its human ligands. These studies will focus on samples of Ail and its complexes incorporated in detergent-free lipid bilayer membranes because the biological activity of Ail is dramatically altered in the presence of micellar detergents. Furthermore, these studies will utilize NMR spectroscopy, a method adept at characterizing both structure and dynamics, because the extracellular loops of Ail that mediate the interactions with host are highly dynamic. Finally, these studies will complement the experimental data generated for Ail in lipid bilayer membranes, by developing computational methods tailored specifically for NMR structural analysis of integral membrane proteins within their functional environment of the phospholipid bilayer membrane. The methods will be designed to facilitate structure determination and increase structural quality, and they will be integrated with widely used programs for programs for NMR structure calculations so as to broaden the impact of our work.